Nucleic acids encoding T2R, a novel family of taste receptors

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of taste cell specific G-protein coupled receptors, antibodies to such receptors, methods of detecting such nucleic acids and receptors, and methods of screening for modulators of taste cell specific G-protein coupled receptors.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to and is a continuation of U.S.Ser. No. 10/364,861 (now U.S. Pat. No. 7,452,694), filed Feb. 10, 2003,which is a continuation-in-part of U.S. Ser. No. 09/393,634 (now U.S.Pat. No. 6,558,910), filed Sep. 10, 1999, the disclosures of which areherein incorporated by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. 5R01DC03160, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences oftaste cell specific G-protein coupled receptors, antibodies to suchreceptors, methods of detecting such nucleic acids and receptors, andmethods of screening for modulators of taste cell specific G-proteincoupled receptors.

BACKGROUND OF THE INVENTION

Taste transduction is one of the most sophisticated forms ofchemotransduction in animals (see, e.g., Margolskee, BioEssays15:645-650 (1993); Avenet & Lindemann, J. Membrane Biol. 112:1-8(1989)). Gustatory signaling is found throughout the animal kingdom,from simple metazoans to the most complex of vertebrates; its mainpurpose is to provide a reliable signaling response to non-volatileligands. Each of these modalities is though to be mediated by distinctsignaling pathways mediated by receptors or channels, leading toreceptor cell depolarization, generation of a receptor or actionpotential, and release of neurotransmitter at gustatory afferent neuronsynapses (see, e.g., Roper, Ann. Rev. Neurosci. 12:329-353 (1989)).

Mammals are believed to have five basic taste modalities: sweet, bitter,sour, salty, and umami (the taste of monosodium glutamate) (see, e.g.,Kawamura & Kare, Introduction to Umami: A Basic Taste (1987); Kinnamon &Cummings, Ann. Rev. Physiol. 54:715-731 (1992); Lindemann, Physiol. Rev.76:718-766 (1996); Stewart et al., Am. J. Physiol. 272:1-26 (1997)).Extensive psychophysical studies in humans have reported that differentregions of the tongue display different gustatory preferences (see,e.g., Hoffmann, Menchen. Arch. Path. Anat. Physiol. 62:516-530 (1875);Bradley et al., Anatomical Record 212: 246-249 (1985); Miller & Reedy,Physiol. Behav. 47:1213-1219 (1990)). Also, numerous physiologicalstudies in animals have shown that taste receptor cells may selectivelyrespond to different tastants (see, e.g., Akabas et al., Science242:1047-1050 (1988); Gilbertson et al., J. Gen. Physiol. 100:803-24(1992); Bernhardt et al., J. Physiol. 490:325-336 (1996); Cummings etal., J. Neurophysiol. 75:1256-1263 (1996)).

In mammals, taste receptor cells are assembled into taste buds that aredistributed into different papillae in the tongue epithelium.Circumvallate papillae, found at the very back of the tongue, containhundreds (mice) to thousands (human) of taste buds and are particularlysensitive to bitter substances. Foliate papillae, localized to theposterior lateral edge of the tongue, contain dozens to hundreds oftaste buds and are particularly sensitive to sour and bitter substances.Fungiform papillae containing a single or a few taste buds are at thefront of the tongue and are thought to mediate much of the sweet tastemodality.

Each taste bud, depending on the species, contains 50-150 cells,including precursor cells, support cells, and taste receptor cells (see,e.g., Lindemann, Physiol. Rev. 76:718-766 (1996)). Receptor cells areinnervated at their base by afferent nerve endings that transmitinformation to the taste centers of the cortex through synapses in thebrain stem and thalamus. Elucidating the mechanisms of taste cellsignaling and information processing is critical for understanding thefunction, regulation, and “perception” of the sense of taste.

Although much is known about the psychophysics and physiology of tastecell function, very little is known about the molecules and pathwaysthat mediate these sensory signaling responses (reviewed by Gilbertson,Current Opin. Neurobiol. 3:532-539 (1993)). Electrophysiological studiessuggest that sour and salty tastants modulate taste cell function bydirect entry of H⁺ and Na⁺ ions through specialized membrane channels onthe apical surface of the cell. In the case of sour compounds, tastecell depolarization is hypothesized to result from H⁺ blockage of K⁺channels (see, e.g., Kinnamon et al., Proc. Nat'l. Acad. Sci. USA 85:7023-7027 (1988)) or activation of pH-sensitive channels (see, e.g.,Gilbertson et al., J. Gen. Physiol. 100:803-24 (1992)); salttransduction may be partly mediated by the entry of Na⁺ viaamiloride-sensitive Na⁺ channels (see, e.g., Heck et al., Science223:403-405 (1984); Brand et al., Brain Res. 207-214 (1985); Avenet etal., Nature 331: 351-354 (1988)).

Sweet, bitter, and umami transduction are believed to be mediated byG-protein-coupled receptor (GPCR) signaling pathways (see, e.g., Striemet al., Biochem. J. 260:121-126 (1989); Chaudhari et al., J. Neuros.16:3817-3826 (1996); Wong et al., Nature 381: 796-800 (1996)).Confusingly, there are almost as many models of signaling pathways forsweet and bitter transduction as there are effector enzymes for GPCRcascades (e.g., G protein subunits, cGMP phosphodiesterase,phospholipase C, adenylate cyclase; see, e.g., Kinnamon & Margolskee,Curr. Opin. Neurobiol. 6:506-513 (1996)). However, little is known aboutthe specific membrane receptors involved in taste transduction, or manyof the individual intracellular signaling molecules activated by theindividual taste transduction pathways. Identification of such moleculesis important given the numerous pharmacological and food industryapplications for bitter antagonists, sweet agonists, and othermodulators of taste.

One taste-cell specific G protein that has been identified is calledGustducin (McLaughin et al., Nature 357:563-569 (1992)). This protein isproposed to be involved in the detection of certain bitter and sweettastes (Wong et al., Nature 381:796-800 (1996)), and is expressed in asignificant subset of cells from all types of taste papillae (McLaughinet al., Nature 357:563-569 (1992)).

Recently, two novel GPCRs were identified and found to be specificallyexpressed in taste cells. While these receptor proteins, called T1R1 andT1R2, appear to be directly involved in taste reception (Hoon et al.,Cell 96:541-551 (1999)), they are only expressed in a fraction ofmammalian taste receptor cells. For example, neither of the genes areextensively expressed in Gustducin-expressing cells. Thus, it is clearthat additional taste-involved GPCRs remain to be discovered.

Genetic studies in mammals have identified numerous loci that areinvolved in the detection of taste. For example, psychophysical tastingstudies have shown that humans can be categorized as tasters,non-tasters, and super-tasters for the bitter substance PROP(6-n-propylthiouracil), and that PROP tasting may be conferred by adominant allele, with non-tasters having two recessive alleles andtasters having at least one dominant allele (see Bartoshuk et al.,Physiol Behav 56(6):1165-71; 58:203-204 (1994)). Recently, a locusinvolved in PROP tasting has been mapped to human interval 5p15 (Reed etal., Am. J. Hum. Genet., 64(5):1478-80 (1999)). The PROP tasting genepresent at the 5p15 locus has yet to be described, however.

In addition, a number of genes involved in taste have been mapped inmice. For example, a cluster of genes involved in bitter-taste detectionhas been mapped to a region of chromosome 6 in mice (Lush et al., GenetRes. 66:167-174 (1995)).

The identification and isolation of novel taste receptors and tastesignaling molecules would allow for new methods of pharmacological andgenetic modulation of taste transduction pathways. For example, theavailability of receptor and channel molecules would permit thescreening for high affinity agonists, antagonists, inverse agonists, andmodulators of taste cell activity. Such taste modulating compounds wouldbe useful in the pharmaceutical and food industries to customize taste.In addition, such taste cell specific molecules can serve as invaluabletools in the generation of taste topographic maps that elucidate therelationship between the taste cells of the tongue and taste sensoryneurons leading to taste centers in the brain.

SUMMARY OF THE INVENTION

The present invention thus provides novel nucleic acids encoding afamily of taste-cell specific G-protein coupled receptors. These nucleicacids and the polypeptides that they encode are referred to as the T2Rfamily of G-protein coupled taste receptors, also as the “SF,” or “GR”family of G-protein coupled taste receptors. This novel family of GPCRsincludes components of the taste transduction pathway. In particular,members of this family are involved in the detection of bitter tastes.

In one aspect, the present invention provides a method for identifying acompound that modulates taste signaling in taste cells, the methodcomprising the steps of: (i) contacting the compound with a tastetransduction G-protein coupled receptor polypeptide, wherein thepolypeptide is expressed in a taste cell, the polypeptide comprisinggreater than about 60% amino acid sequence identity to a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80; and (ii)determining the functional effect of the compound upon the polypeptide.

In another aspect, the present invention provides a method foridentifying a compound that modulates taste signaling in taste cells,the method comprising the steps of: (i) contacting the compound with apolypeptide comprising an extracellular domain of a taste transductionG-protein coupled receptor, wherein the receptor is expressed in a tastecell, the extracellular domain comprising greater than about 60% aminoacid sequence identity to the extracellular domain of a polypeptidecomprising a sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ IDNO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ IDNO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80; and(ii) determining the functional effect of the compound upon theextracellular domain.

In another aspect, the present invention provides a method foridentifying a compound that modulates taste signaling in taste cells,the method comprising the steps of: (i) contacting the compound with ataste transduction G-protein coupled receptor polypeptide comprisingeither: (a) a sequence comprising at least about 50% amino acid identityto a sequence selected from the group consisting of SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87; or(b) a sequence selected from the group consisting of SEQ ID NO:88, SEQID NO:89, SEQ ID NO:90, and SEQ ID NO:91; and (ii) determining thefunctional effect of the compound upon the polypeptide.

In one embodiment, the polypeptide has G-protein coupled receptoractivity. In another embodiment, the functional effect of the compoundupon the polypeptide is determined by measuring changes in intracellularcAMP, cGMP, IP3, or Ca²⁺. In another embodiment, the functional effectis a chemical effect. In another embodiment, the functional effect is aphysical effect. In another embodiment, the functional effect isdetermined by measuring binding of the compound to an extracellulardomain of the polypeptide. In another embodiment, the functional effectis determined by measuring radiolabeled GTP binding to the polypeptide.In another embodiment, the functional effect is measured by determiningchanges in the electrical activity of cells expressing the polypeptides.

In another embodiment, the polypeptide or parts thereof is recombinant.In another embodiment, the polypeptide comprises an extracellular domainthat is covalently linked to a heterologous polypeptide, forming achimeric polypeptide. In another embodiment, the polypeptide is linkedto a solid phase, either covalently or non-covalently.

In another embodiment, the polypeptide is from a rat, a mouse, or ahuman. In another embodiment, the polypeptide is expressed in a cell ora cell membrane. In another embodiment, the cell is a eukaryotic cell.In another embodiment, the polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ BD NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80.

In one aspect, the present invention provides an isolated nucleic acidencoding a taste transduction G-protein coupled receptor, wherein thereceptor is expressed in a taste cell, the receptor comprising greaterthan about 60% amino acid sequence identity to a sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33.

In another aspect, the present invention provides an isolated nucleicacid encoding a taste transduction G-protein coupled receptor, whereinthe nucleic acid specifically hybridizes under highly stringentconditions to a nucleic acid having a nucleotide sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10; SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34, but not to anucleic acid having a nucleotide sequence selected from the groupconsisting of SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46; SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73,SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, and SEQ ID NO:81.

In another aspect, the present invention provides an isolated nucleicacid encoding a taste transduction G-protein coupled receptor, thereceptor comprising greater than about 60% amino acid identity to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, and SEQ ID NO:33, wherein the nucleic acidselectively hybridizes under moderately stringent hybridizationconditions to a nucleotide sequence having a nucleotide sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10; SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34 butnot to a nucleic acid having a nucleotide sequence selected from thegroup consisting of SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46; SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, and SEQ ID NO:81.

In another aspect, the present invention provides an isolated nucleicacid encoding an extracellular domain of a taste transduction G-proteincoupled receptor, wherein the receptor is expressed in a taste cell, theextracellular domain having greater than about 60% amino acid sequenceidentity to the extracellular domain of a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQID NO:33.

In one embodiment, the nucleic acid encodes a receptor that specificallybinds to polyclonal antibodies generated against a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQID NO:33, but not to polyclonal antibodies generated against apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70,SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ IDNO:80.

In another embodiment, the nucleic acid encodes a receptor comprising anamino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33.

In another embodiment, the nucleic acid comprises a nucleotide sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10; SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34.

In another embodiment, the nucleic acid encodes a receptor that hasG-protein coupled receptor activity. In another embodiment, the nucleicacid is from a rat or a mouse. In another embodiment, the nucleic acidis amplified by primers that selectively hybridize under stringenthybridization conditions to the same sequence as degenerate primer setsencoding amino acid sequences selected from the group consisting of:KMAPLDLLL (SEQ ID NO:88), ATWLGVFYCAK (SEQ ID NO:89), LSILT2RLILY (SEQID NO:90), and LILGNPKLK (SEQ ID NO:91).

In one embodiment, the nucleic acid encodes the extracellular domainlinked to a heterologous polypeptide, forming a chimeric polypeptide. Inanother embodiment, the nucleic acid encodes the extracellular domain ofa polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, and SEQ ID NO:33.

In another aspect, the present invention provides an isolated tastetransduction G-protein coupled receptor, wherein the receptor isexpressed in a taste cell, the receptor comprising greater than about60% amino acid sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, and SEQ ID NO:33.

In one embodiment, the receptor specifically binds to polyclonalantibodies generated against a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33, butnot to polyclonal antibodies generated against a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:35,SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45,SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64,SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74,SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80. In another embodiment, thereceptor has G-protein coupled receptor activity. In another embodiment,the receptor has an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, and SEQ ID NO:33. In another embodiment, thereceptor is from a rat or a mouse.

In one aspect, the present invention provides an isolated polypeptidecomprising an extracellular domain of a taste transduction G-proteincoupled receptor, wherein the receptor is expressed in a taste cell, theextracellular domain comprising greater than about 60% amino acidsequence identity to the extracellular domain of a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, and SEQ ID NO:33.

In one embodiment, the polypeptide encodes the extracellular domain of apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, and SEQ ID NO:33. In another embodiment, theextracellular domain is covalently linked to a heterologous polypeptide,forming a chimeric polypeptide.

In one aspect, the present invention provides an antibody thatselectively binds to the receptor comprising greater than about 60%amino acid sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, and SEQ ID NO:33.

In another aspect, the present invention provides an expression vectorcomprising a nucleic acid encoding a taste transduction G-proteincoupled receptor, wherein the receptor is expressed in a taste cell, thereceptor comprising greater than about 60% amino acid sequence identityto a sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ IDNO:33.

In another aspect, the present invention provides a host celltransfected with the expression vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides nucleotide sequence, amino acid sequence, and geneticdata for various rat, mouse, and human T2R family members.

The polypeptide and polynucleotide sequences of rGR01 are SEQ ID NOs:1and 2, respectively.

The polypeptide and polynucleotide sequences of rGR02 are SEQ ID NOs:3and 4, respectively.

The polypeptide and polynucleotide sequences of rGR03 are SEQ ID NOs:5and 6, respectively.

The polypeptide and polynucleotide sequences of rGR04 are SEQ ID NOs:7and 8, respectively.

The polypeptide and polynucleotide sequences of rGR05 are SEQ ID NOs:9and 10, respectively.

The polypeptide and polynucleotide sequences of mGR01 are SEQ ID NOs:11and 12, respectively.

The polypeptide and polynucleotide sequences of mGR02 are SEQ ID NOs:13and 14, respectively.

The polypeptide and polynucleotide sequences of mGR03 are SEQ ID NOs: 15and 16, respectively.

The polypeptide and polynucleotide sequences of mGR04 are SEQ ID NOs: 17and 18, respectively.

The polypeptide and polynucleotide sequences of mGR05 are SEQ ID NOs:19and 20, respectively.

The polypeptide and polynucleotide sequences of mGR06 are SEQ ID NOs:21and 22, respectively.

The polypeptide and polynucleotide sequences of mGR07 are SEQ ID NOs:23and 24, respectively.

The polypeptide and polynucleotide sequences of mGR08 are SEQ ID NOs:25and 26, respectively.

The polypeptide and polynucleotide sequences of mGR09 are SEQ ID NOs:27and 28, respectively.

The polypeptide and polynucleotide sequences of mGR10 are SEQ ID NOs:29and 30, respectively.

The polypeptide and polynucleotide sequences of mGR 11 are SEQ ID NOs:31and 32, respectively.

The polypeptide and polynucleotide sequences of mGR13 are SEQ ID NOs:33and 34, respectively.

The polypeptide and polynucleotide sequences of hGR01 are SEQ ID NOs:35and 36, respectively.

The polypeptide and polynucleotide sequences of hGR02 are SEQ ID NOs:37and 38, respectively.

The polypeptide and polynucleotide sequences of hGR03 are SEQ ID NOs:39and 40, respectively.

The polypeptide and polynucleotide sequences of hGR04 are SEQ ID NOs:41and 42, respectively.

The polypeptide and polynucleotide sequences of hGR05 are SEQ ID NOs:43and 44, respectively.

The polypeptide and polynucleotide sequences of hGR06 are SEQ ID NOs:45and 46, respectively.

The polypeptide and polynucleotide sequences of hGR07 are SEQ ID NOs:47and 48, respectively.

The polypeptide and polynucleotide sequences of hGR08 are SEQ ID NOs:49and 50, respectively.

The polypeptide and polynucleotide sequences of hGR09 are SEQ ID NOs:51and 52, respectively.

The polypeptide and polynucleotide sequences of hGR10 are SEQ ID NOs:53and 54, respectively.

The polypeptide and polynucleotide sequences of hGR11 are SEQ ID NOs:55and 56., respectively.

The polypeptide and polynucleotide sequences of hGR12 are SEQ ID NOs:57and 58, respectively.

The polypeptide and polynucleotide sequences of hGR13 are SEQ ID NOs:59and 60, respectively.

The polypeptide and polynucleotide sequences of hGR14 are SEQ ID NOs:61and 62, respectively.

The polypeptide and polynucleotide sequences of hGR15 are SEQ ID NOs:63and 64, respectively.

The polypeptide and polynucleotide sequences of hGR16 are SEQ ID NOs:65and 66, respectively.

The polypeptide and polynucleotide sequences of hGR17 are SEQ ID NOs:67and 68, respectively.

The polypeptide and polynucleotide sequences of hGR18 are SEQ ID NOs:69and 70, respectively.

The polypeptide and polynucleotide sequences of hGR19 are SEQ ID NOs:71and 72, respectively.

The polypeptide and polynucleotide sequences of hGR20 are SEQ ID NOs:73and 74, respectively.

The polypeptide and polynucleotide sequences of hGR21 are SEQ ID NOs:75and 76, respectively.

The polypeptide and polynucleotide sequences of hGR22 are SEQ ID NOs:77and 78, respectively.

The polypeptide and polynucleotide sequences of hGR23 are SEQ ID NOs:79and 80, respectively.

The polypeptide and polynucleotide sequences of hGR24 are SEQ ID NOs:81and 82, respectively.

FIG. 2 provides a dendogram showing the relationship between some of thevarious T2R family members.

FIG. 3 provides a comparison of amino acid sequences of some of thevarious T2R family members. The SEQ ID NOs of the disclosed sequencesare listed above for FIG. 1.

FIG. 4 a-b: (a) Structure of T2R5 and (b) Southern blot demonstratinghomologous recombination and disruption of the T2R5 gene.

FIG. 5 a-c: T2R5 animals cannot taste the bitter tastant cycloheximidebut respond normally to other bitter tastants (e.g. quinine, PROP,denatonium). Control animals display strong aversive responses to allbitter tastants, including cycloheximide.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides nucleic acids encoding a novel family oftaste cell specific G-protein coupled receptors. These nucleic acids andthe receptors that they encode are referred to as members of the “SF” or“T2R” family of taste cell specific G protein coupled receptors. Thesetaste cell specific GPCRs are components of the taste transductionpathway, and are involved in the taste detection of substances such asthe bitter substances denatonium, 6-n-propylthiouracil (PROP), sucroseoctaacetate (soa), ruffinose acetate (roa), cycloheximide (cyx), andquinine (qui). These nucleic acids provide valuable probes for theidentification of taste cells, as the nucleic acids are specificallyexpressed in taste cells. For example, probes for T2R polypeptides andproteins can be used to identity subsets of taste cells such as foliatecells and circumvallate cells, or specific taste receptor cells, e.g.,sweet, sour, salty, and bitter. They also serve as tools for thegeneration of taste topographic maps that elucidate the relationshipbetween the taste cells of the tongue and taste sensory neurons leadingto taste centers in the brain. Furthermore, the nucleic acids and theproteins they encode can be used as probes to dissect taste-inducedbehaviors.

The invention also provides methods of screening for modulators, e.g.,activators, inhibitors, stimulators, enhancers, agonists, andantagonists, of these novel taste cell GPCRs. Such modulators of tastetransduction are useful for pharmacological and genetic modulation oftaste signaling pathways. These methods of screening can be used toidentify high affinity agonists and antagonists of taste cell activity.These modulatory compounds can then be used in the food andpharmaceutical industries to customize taste. Thus, the inventionprovides assays for taste modulation, where members of the T2R familyact as direct or indirect reporter molecules for the effect ofmodulators on taste transduction. GPCRs can be used in assays, e.g., tomeasure changes in ligand binding, ion concentration, membranepotential, current flow, ion flux, transcription, signal transduction,receptor-ligand interactions, second messenger concentrations, in vitro,in vivo, and ex vivo. In one embodiment, members of the T2R family canbe used as indirect reporters via attachment to a second reportermolecule such as green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961-964 (1997)). In another embodiment,T2R family members are recombinantly expressed in cells, and modulationof taste transduction via GPCR activity is assayed by measuring changesin Ca²⁺ levels and other intracellular messages such as cAMP, cGMP, andIP3.

Methods of assaying for modulators of taste transduction include invitro ligand binding assays using T2R polypeptides, portions thereofsuch as the extracellular domain, or chimeric proteins comprising one ormore domains of a T2R family member, oocyte T2R gene expression; tissueculture cell T2R gene expression; transcriptional activation of T2Rgenes; phosphorylation and dephosphorylation of T2R family members;G-protein binding to GPCRs; ligand binding assays; voltage, membranepotential and conductance changes; ion flux assays; changes inintracellular second messengers such as cGMP, cAMP and inositoltriphosphate; changes in intracellular calcium levels; andneurotransmitter release.

Finally, the invention provides methods of detecting T2R nucleic acidand protein expression, allowing investigation of taste transductionregulation and specific identification of taste receptor cells. T2Rfamily members also provide useful nucleic acid probes for paternity andforensic investigations. T2R genes are also useful as a nucleic acidprobe for identifying subpopulations of taste receptor cells such asfoliate, fungiform, and circumvallate taste receptor cells. T2Rreceptors can also be used to generate monoclonal and polyclonalantibodies useful for identifying taste receptor cells. Taste receptorcells can be identified using techniques such as reverse transcriptionand amplification of mRNA, isolation of total RNA or poly A⁺ RNA,northern blotting, dot blotting, in situ hybridization, RNaseprotection, S1 digestion, probing DNA microchip arrays, western blots,and the like.

The T2R genes comprise a large family of related taste cell specificG-multiple protein coupled receptors. Within the genome, these genes arepresent either alone or within one of several gene clusters. One genecluster, located at human genomic region 12p13, comprises at least 9genes, and a second cluster, located at 7q31, comprises at least 4genes. In total, 24 distinct T2R family members have been identified,including several putative pseudogenes.

Further, some of the T2R genes are associated with previously mappedmammalian taste-specific loci. For example, the human SF01 is located athuman interval 5p15, precisely where the locus underlying the ability totaste the substance PROP has previously been mapped. In addition, thehuman gene cluster found at genomic region 12p13 corresponds to a regionof mouse chromosome 6 that has been shown to contain numerousbitter-tasting genes, including sucrose octaacetate, ruffinose acetate,cycloheximide, and quinine (see, e.g., Lush et al., Genet. Res.6:167-174 (1995)). These associations indicate that the T2R genes areinvolved in the taste detection of various substances, in particularbitter substances.

Functionally, the T2R genes comprise a family of related seventransmembrane G-protein coupled receptors involved in tastetransduction, which interact with a G-protein to mediate taste signaltransduction (see, e.g., Fong, Cell Signal 8:217 (1996); Baldwin, Curr.Opin. Cell Biol. 6:180 (1994)).

Structurally, the nucleotide sequence of T2R family members (see, e.g.,SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, and 81, isolated from rats, mice, and humans)encodes a family of related polypeptides comprising an extracellulardomain, seven transmembrane domains, and a cytoplasmic domain. RelatedT2R family genes from other species share at least about 60% nucleotidesequence identity over a region of at least about 50 nucleotides inlength, optionally 100, 200, 500, or more nucleotides in length, to SEQID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, or 81, or encode polypeptides sharing at least about 60%amino acid sequence identity over an amino acid region at least about 25amino acids in length, optionally 50 to 100 amino acids in length to SEQID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, or 80. T2R genes are specifically expressed in tastecells.

Several consensus amino acid sequences have also been discovered thatare characteristic of T2R family members. For example, T2R familymembers typically comprise a sequence at least about 50% identical toSEQ ID NO:82 (corresponding, e.g., to amino acid positions 16-35 in SEQID NOS:1 and 35, see also FIG. 3, transmembrane region 1), 83(corresponding, e.g., to amino acid positions 45-58 in SEQ ID NOS:1 and35, see also FIG. 3, transmembrane region 2), 84 (corresponding, e.g.,to amino acid positions 89-101 in SEQ ID NOS:1 and 35, see also FIG. 3,transmembrane region 3), 85 (corresponding, e.g., to amino acidpositions 102-119 in SEQ ID NOS:1 and 35, see also FIG. 3, transmembraneregion 3), 86 (corresponding, e.g., to amino acid positions 195-208 inSEQ ID NO:1, and to amino acid positions 196-209 in SEQ ID NO:35, seealso FIG. 3, transmembrane region 5), or 87 (corresponding, e.g., toamino acid positions 271-284 in SEQ ID NO:1, and to amino acid positions273-286 in SEQ ID NO:35, see also FIG. 3, transmembrane region 7).

One T2R gene, SF01, has been identified in numerous species, includingin rats (SEQ ID NOS:1, 2 for amino acid and nucleotide sequence,respectively) and humans (SEQ ID NO:35, 36 for amino acid and nucleotidesequence, respectively), and can be defined according to one or moreSF01 (also referred to as GR01) signature sequences. Accordingly, GR01polypeptides typically comprise an amino acid sequence shown as SEQ IDNO:88 (corresponding, e.g., to amino acid positions 40-48 in SEQ ID NOS:1 and 35), 89 (corresponding, e.g., to amino acid positions 96-106 inSEQ ID NOS: 1 and 35), 90 (corresponding, e.g., to amino acid positions226-235 in SEQ ID NO: 1, and to positions 228-237 in SEQ ID NO: 35), or91 (corresponding, e.g., to amino acid positions 275-283 in SEQ ID NO:1, and to positions 277-285 in SEQ ID NO: 35).

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:1: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 7;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 20.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:3: variant #1, in which a tyrosine residueis substituted for a phenylalanine residue at amino acid position 2; andvariant #2, in which a valine residue is substituted for an isoleucineresidue at amino acid position 62.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:5: variant #4; and variant #2, in which aleucine residue is substituted for an isoleucine residue at amino acidposition 64.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:9: variant #1, in which a valine residueis substituted for an isoleucine residue at amino acid position 56; andvariant #2, in which a methionine residue is substituted for a cysteineresidue at amino acid position 57.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:11: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 2;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 7.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:13: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 2;and variant #2, in which an isoleucine residue is substituted for avaline residue at amino acid position 5.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:15: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 61;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 68.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:17: variant #1, in which a glycine residueis substituted for an alanine residue at amino acid position 4; andvariant #2, in which a phenylalanine residue is substituted for atryptophan residue at amino acid position 60.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:19: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 62;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 244.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:21: variant #1, in which a serine residueis substituted for a threonine residue at amino acid position 3; andvariant #2, in which a lysine residue is substituted for an arginineresidue at amino acid position 123.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:23: variant #1, in which an asparagineresidue is substituted for a glutamine residue at amino acid position63; and variant #2, in which a leucine residue is substituted for anisoleucine residue at amino acid position 59.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:25: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 2;and variant #2, in which an aspartic acid residue is substituted for aglutamic acid residue at amino acid position 4.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:27: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 16;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 46.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:29: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 9;and variant #2 in which a tryptophan residue is substituted for aphenylalanine residue at amino acid position 14.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:31: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 5;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 60.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:33: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 60;and variant #2, in which a histidine residue is substituted for a lysineresidue at amino acid position 65.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:35: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 6;and variant #2, in which a glycine residue is substituted for an alanineresidue at amino acid position 13.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:37: variant #1, in which a leucine residueis substituted for an isoleucine residue at amino acid position 11; andvariant #2, in which a threonine residue is substituted for a serineresidue at amino acid position 15.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:39: variant #1, in which an isoleucineresidue is substituted for a valine residue at amino acid position 8;and variant #2, in which an asparagine residue is substituted for aglutamine residue at amino acid position 16.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:41: variant #1, in which a lysine residueis substituted for an arginine residue at amino acid position 3; andvariant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 20.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:43: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 6;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 23.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:45: variant #1, in which a leucine residueis substituted for an isoleucine residue at amino acid position 12; andvariant #2, in which a aspartic acid residue is substituted for aglutamic acid residue at amino acid position 16.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:47: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 10;and variant #2, in which a glycine residue is substituted for an alanineresidue at amino acid position 25.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:49: variant #1, in which a tryptophanresidue is substituted for a phenylalanine residue at amino acidposition 9; and variant #2, in which an alanine residue is substitutedfor a glycine residue at amino acid position 25.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:51: variant #1, in which a serine residueis substituted for a threonine residue at amino acid position 18; andvariant #2, in which a leucine residue is substituted for an isoleucineresidue at amino acid position 33.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:53: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 2;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 7.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:55: variant 190 1, in which an arginineresidue is substituted for a lysine residue at amino acid position 6;and variant #2, in which a leucine residue is substituted for a valineresidue at amino acid position 26.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:56: variant #1, in which a leucine residueis substituted for an isoleucine residue at amino acid position 4; andvariant #2, in which a lysine residue is substituted for an arginineresidue at amino acid position 11.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:58: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 37;and variant #2, in which a glutamic acid residue is substituted for anaspartic acid residue at amino acid position 45.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:60: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 61;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 123.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:62: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 5;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 57.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:64: variant #1, in which a serine residueis substituted for a threonine residue at amino acid position 182; andvariant #2, in which an isoleucine residue is substituted for a leucineresidue at amino acid position 185.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:66: variant #1, in which an alanineresidue is substituted for a glycine residue at amino acid position 14;and variant #2, in which a phenylalanine residue is substituted for atryptophan residue at amino acid position 60.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:68: variant #1, in which a leucine residueis substituted for an isoleucine residue at amino acid position 5; andvariant #2, in which a glycine residue is substituted for an alanineresidue at amino acid position 13.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:70: variant #1, in which a glycine residueis substituted for an alanine residue at amino acid position 61; andvariant #2, in which a valine residue is substituted for a leucineresidue at amino acid position 65.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:72: variant #1, in which a lysine residueis substituted for an arginine residue at amino acid position 4; andvariant #2, in which a leucine residue is substituted for a valineresidue at amino acid position 60.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:74: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 5;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 53.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:76: variant #1, in which a glutamic acidresidue is substituted for an aspartic acid residue at amino acidposition 6; and variant #2, in which an isoleucine residue issubstituted for a leucine residue at amino acid position 63.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:78: variant #1, in which an isoleucineresidue is substituted for a valine residue at amino acid position 4;and variant #2, in which a glycine residue is substituted for an alanineresidue at amino acid position 9.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:80: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 5;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 57.

Specific regions of the T2R nucleotide and amino acid sequences may beused to identify polymorphic variants, interspecies homologs, andalleles of T2R family members. This identification can be made in vitro,e.g., under stringent hybridization conditions or PCR (e.g., usingprimers encoding SEQ ID NOS:88-91) and sequencing, or by using thesequence information in a computer system for comparison with othernucleotide sequences. Typically, identification of polymorphic variantsand alleles of T2R family members is made by comparing an amino acidsequence of about 25 amino acids or more, e.g., 50-100 amino acids.Amino acid identity of approximately at least 60% or above, optionally65%, 70%, 75%, 80%, 85%, or 90-95% or above typically demonstrates thata protein is a polymorphic variant, interspecies homolog, or allele of aT2R family member. Sequence comparison can be performed using any of thesequence comparison algorithms discussed below. Antibodies that bindspecifically to T2R polypeptides or a conserved region thereof can alsobe used to identify alleles, interspecies homologs, and polymorphicvariants.

Polymorphic variants, interspecies homologs, and alleles of T2R genesare confirmed by examining taste cell specific expression of theputative T2R polypeptide. Typically, T2R polypeptides having an aminoacid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, or SEQ ID NO:80 is used as a positive control in comparison tothe putative T2R protein to demonstrate the identification of apolymorphic variant or allele of the T2R family member. The polymorphicvariants, alleles and interspecies homologs are expected to retain theseven transmembrane structure of a G-protein coupled receptor.

Nucleotide and amino acid sequence information for T2R family membersmay also be used to construct models of taste cell specific polypeptidesin a computer system. These models are subsequently used to identifycompounds that can activate or inhibit T2R receptor proteins. Suchcompounds that modulate the activity of T2R family members can be usedto investigate the role of T2R genes in taste transduction.

The isolation of T2R family members provides a means for assaying forinhibitors and activators of G-protein coupled receptor tastetransduction. Biologically active T2R proteins are useful for testinginhibitors and activators of T2R as taste transducers using in vivo andin vitro assays that measure, e.g., transcriptional activation of T2R;ligand binding; phosphorylation and dephosphorylation; binding toG-proteins; G-protein activation; regulatory molecule binding; voltage,membrane potential and conductance changes; ion flux; intracellularsecond messengers such as cGMP, cAMP and inositol triphosphate;intracellular calcium levels; and neurotransmitter release. Suchactivators and inhibitors identified using T2R family members can beused to further study taste transduction and to identify specific tasteagonists and antagonists. Such activators and inhibitors are useful aspharmaceutical and food agents for customizing taste.

The present invention also provides assays, preferably high throughputassays, to identify molecules that interact with and/or modulate a T2Rpolypeptide. In numerous assays, a particular domain of a T2R familymember is used, e.g., an extracellular, transmembrane, or intracellulardomain. In numerous embodiments, an extracellular domain is bound to asolid substrate, and used, e.g., to isolate ligands, agonists,antagonists, or any other molecule that can bind to and/or modulate theactivity of an extracellular domain of a T2R polypeptide. In certainembodiments, a domain of a T2R polypeptide, e.g., an extracellular,transmembrane, or intracellular domain, is fused to a heterologouspolypeptide, thereby forming a chimeric polypeptide, e.g., a chimericpolypeptide with G protein coupled receptor activity. Such chimericpolypeptides are useful, e.g., in assays to identify ligands, agonists,antagonists, or other modulators of a T2R polypeptide. In addition, suchchimeric polypeptides are useful to create novel taste receptors withnovel ligand binding specificity, modes of regulation, signaltransduction pathways, or other such properties, or to create noveltaste receptors with novel combinations of ligand binding specificity,modes of regulation, signal transduction pathways, etc.

Methods of detecting T2R nucleic acids and expression of T2Rpolypeptides are also useful for identifying taste cells and creatingtopological maps of the tongue and the relation of tongue taste receptorcells to taste sensory neurons in the brain. Chromosome localization ofthe genes encoding human T2R genes can be used to identify diseases,mutations, and traits caused by and associated with T2R family members.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Taste cells” include neuroepithelial cells that are organized intogroups to form taste buds of the tongue, e.g., foliate, fungiform, andcircumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci.12:329-353 (1989)). Taste cells also include cells of the palate, andother tissues that may contain taste cells such as the esophagus and thestomach.

“SF” or “T2R” refers to one or more members of a family of G-proteincoupled receptors that are expressed in taste cells such as foliate,fungiform, and circumvallate cells, as well as cells of the palate,esophagus, and stomach (see, e.g., Hoon et al., Cell 96:541-551 (1999);Chandrashekar et al., Cell 100:703-711 (2000); Adler et al., Cell100:693-702 (2000); Bufe et al., Nature Genetics 32:397-401 (2002), andWO 01/77676, herein each incorporated by reference in their entirety).Such taste cells can be identified because they express specificmolecules such as Gustducin, a taste cell specific G protein, or othertaste specific molecules (McLaughin et al., Nature 357:563-569 (1992)).Taste receptor cells can also be identified on the basis of morphology(see, e.g., Roper, supra). T2R family members have the ability to act asreceptors for taste transduction. T2R family members are also referredto as the “GR” family, for gustatory receptor.

“SF” or “T2R” nucleic acids encode a family of GPCRs with seventransmembrane regions that have “G-protein coupled receptor activity,”e.g., they bind to G-proteins in response to extracellular stimuli andpromote production of second messengers such as IP3, cAMP, cGMP, andCa²⁺ via stimulation of enzymes such as phospholipase C and adenylatecyclase (for a description of the structure and function of GPCRs, see,e.g., Fong, supra, and Baldwin, supra). A dendogram providing therelationship between certain T2R family members is provided as FIG. 2.These nucleic acids encode proteins that are expressed in taste cells,such as

The term “SF” or “T2R” family therefore refers to polymorphic variants,alleles, mutants, and interspecies homologs that: (1) have about 60%amino acid sequence identity, optionally about 75, 80, 85, 90, or 95%amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5;SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, or SEQ ID NO:80 over a window of about 25 aminoacids, optionally 50-100 amino acids; (2) specifically bind toantibodies raised against an immunogen comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80, and conservativelymodified variants thereof; (3) specifically hybridize (with a size of atleast about 100, optionally at least about 500-1000 nucleotides) understringent hybridization conditions to a sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, and SEQID NO:81, and conservatively modified variants thereof; (4) comprise asequence at least about 50% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84,SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:53; or (5) are amplified byprimers that specifically hybridize under stringent hybridizationconditions to the same sequence as a degenerate primer sets encoding SEQID NOS:88, 89, 90, or 91.

SF01, or GR01, refers to a specific member of the T2R family that hasbeen identified in rat (SEQ ID NOS:1, 2), mouse (SEQ ID NO:11, 12), andhuman (SEQ ID NOS:35, 36). Accordingly, “T2R01,” “SFR01,” or “GR01”refers to a nucleic acid comprising a sequence comprising at least about60%, 65%, 70%, 80%, 85%, 90-95%, or more nucleotide sequence identity toSEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:36, or to a polypeptidecomprising an amino acid sequence at least about 60%, 65%, 70%, 80%,85%, 90-95%, or more identical to SEQ ID NO:1, SEQ ID NO:11, or SEQ IDNO:35, or comprising an amino acid sequence at least about 90%, 95%,99%, or more amino acid sequence identity to SEQ ID NO:88, SEQ ID NO:89,SEQ ID NO:90, or SEQ ID NO:91.

Topologically, sensory GPCRs have an “N-terminal domain” “extracellulardomains,” a “transmembrane domain” comprising seven transmembraneregions, cytoplasmic, and extracellular loops, “cytoplasmic domains,”and a “C-terminal domain” (see, e.g., Hoon et al., Cell 96:541-551(1999); Buck & Axel, Cell 65:175-187 (1991)). These domains can bestructurally identified using methods known to those of skill in theart, such as sequence analysis programs that identify hydrophobic andhydrophilic domains (see, e.g., Stryer, Biochemistry (3^(rd) ed. 1988);see also any of a number of Internet based sequence analysis programs,such as those found at dot.imgen.bcm.tmc.edu). Such domains are usefulfor making chimeric proteins and for in vitro assays of the invention,e.g., ligand binding assays.

“Extracellular domains” therefore refers to the domains of T2Rpolypeptides that protrude from the cellular membrane and are exposed tothe extracellular face of the cell. Such domains would include the “Nterminal domain” that is exposed to the extracellular face of the cell,as well as the extracellular loops of the transmembrane domain that areexposed to the extracellular face of the cell, i.e., the loops betweentransmembrane regions 2 and 3, and between transmembrane regions 4 and5. The “N terminal domain” region starts at the N-terminus and extendsto a region close to the start of the transmembrane domain. Theseextracellular domains are useful for in vitro ligand binding assays,both soluble and solid phase.

“Transmembrane domain,” which comprises the seven transmembrane regions,refers to the domain of T2R polypeptides that lies within the plasmamembrane, and may also include the corresponding cytoplasmic(intracellular) and extracellular loops. The seven transmembrane regionscan be identified using standard methods, as described in Kyte &Doolittle, J. Mol. Biol. 157:105-132 (1982)), or in Stryer, supra.

“Cytoplasmic domains” refers to the domains of T2R proteins that facethe inside of the cell, e.g., the “C terminal domain” and theintracellular loops of the transmembrane domain, e.g., the intracellularloops between transmembrane regions 1 and 2, and the intracellular loopsbetween transmembrane regions 3 and 4. “C terminal domain” refers to theregion that spans the end of the last transmembrane domain and theC-terminus of the protein, and which is normally located within thecytoplasm.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains one or more T2R nucleic acids encoding one or moreT2R proteins. Such samples include, but are not limited to, tissueisolated from humans, mice, and rats, in particular, tongue, palate, andother tissues that may contain taste cells such as the esophagus and thestomach. Biological samples may also include sections of tissues such asfrozen sections taken for histological purposes. A biological sample istypically obtained from a eukaryotic organism, such as insects,protozoa, birds, fish, reptiles, and preferably a mammal such as rat,mouse, cow, dog, guinea pig, or rabbit, and most preferably a primatesuch as chimpanzees or humans. Tissues include tongue tissue, isolatedtaste buds, and testis tissue.

“GPCR activity” refers to the ability of a GPCR to transduce a signal.Such activity can be measured in a heterologous cell, by coupling a GPCR(or a chimeric GPCR) to either a G-protein or promiscuous G-protein suchas Gα15, and an enzyme such as PLC, and measuring increases inintracellular calcium using (Offermans & Simon, J. Biol. Chem.270:15175-15180 (1995)). Receptor activity can be effectively measuredby recording ligand-induced changes in [Ca²⁺]_(i) using fluorescentCa²⁺-indicator dyes and fluorometric imaging. Optionally, thepolypeptides of the invention are involved in sensory transduction,optionally taste transduction in taste cells.

The phrase “functional effects” in the context of assays for testingcompounds that modulate T2R family member mediated taste transductionincludes the determination of any parameter that is indirectly ordirectly under the influence of the receptor, e.g., functional, physicaland chemical effects. It includes ligand binding, changes in ion flux,membrane potential, current flow, transcription, G-protein binding, GPCRphosphorylation or dephosphorylation, signal transduction,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP, IP3, or intracellular Ca²⁺), in vitro, in vivo, and ex vivoand also includes other physiologic effects such increases or decreasesof neurotransmitter or hormone release.

By “determining the functional effect” is meant assays for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a T2R family member, e.g., functional, physicaland chemical effects. Such functional effects can be measured by anymeans known to those skilled in the art, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility properties,patch clamping, voltage-sensitive dyes, whole cell currents,radioisotope efflux, inducible markers, oocyte T2R gene expression;tissue culture cell T2R expression; transcriptional activation of T2Rgenes; ligand binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cAMP, cGMP, and inositol triphosphate (IP3); changesin intracellular calcium levels; neurotransmitter release, and the like.

“Inhibitors,” “activators,” and “modulators” of T2R genes or proteinsare used interchangeably to refer to inhibitory, activating, ormodulating molecules identified using in vitro and in vivo assays fortaste transduction, e.g., ligands, agonists, antagonists, and theirhomologs and mimetics. Inhibitors are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate tastetransduction, e.g., antagonists. Activators are compounds that, e.g.,bind to, stimulate, increase, open, activate, facilitate, enhanceactivation, sensitize or up regulate taste transduction, e.g., agonists.Modulators include compounds that, e.g., alter the interaction of areceptor with: extracellular proteins that bind activators or inhibitor(e.g., ebnerin and other members of the hydrophobic carrier family);G-proteins; kinases (e.g., homologs of rhodopsin kinase and betaadrenergic receptor kinases that are involved in deactivation anddesensitization of a receptor); and arrestin-like proteins, which alsodeactivate and desensitize receptors. Modulators include geneticallymodified versions of T2R family members, e.g., with altered activity, aswell as naturally occurring and synthetic ligands, antagonists,agonists, small chemical molecules and the like. Such assays forinhibitors and activators include, e.g., expressing T2R family membersin cells or cell membranes, applying putative modulator compounds, andthen determining the functional effects on taste transduction, asdescribed above. Samples or assays comprising T2R family members thatare treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of inhibition. Control samples(untreated with inhibitors) are assigned a relative T2R activity valueof 100%. Inhibition of a T2R is achieved when the T2R activity valuerelative to the control is about 80%, optionally 50% or 25-0%.Activation of a T2R is achieved when the T2R activity value relative tothe control is 110%, optionally 150%, optionally 200-500%, or 1000-3000%higher.

“Biologically active” T2R refers to a T2R having GPCR activity asdescribed above, involved in taste transduction in taste receptor cells.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolated T2Rnucleic acid is separated from open reading frames that flank the T2Rgene and encode proteins other than a T2R. The term “purified” denotesthat a nucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, optionally at least 95% pure, andoptionally at least 99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)    (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 50 to 350 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are optionally directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Optionally, the identity exists over aregion that is at least about 50 amino acids or nucleotides in length,or more preferably over a region that is 75-100 amino acids ornucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat.'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment (see, e.g., FIG. 2). PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984)).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al, J Mol. Biol. 215:403-410 (1990),respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information (Onthe World Wide Web at ncbi.nlm.nih.gov). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (B) of 10, M5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-T2R” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by a T2R gene, cDNA, or asubsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to a T2R family member from specific species such as rat, mouse,or human can be selected to obtain only those polyclonal antibodies thatare specifically immunoreactive with the T2R protein and not with otherproteins, except for polymorphic variants and alleles of the T2Rprotein. This selection may be achieved by subtracting out antibodiesthat cross-react with T2R molecules from other species. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988), for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, e.g., cultured cells, explants, and cells in vivo.

III. Isolation of Nucleic Acids Encoding T2R Family Members

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter etal., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingT2R Family Members

In general, the nucleic acid sequences encoding T2R family members andrelated nucleic acid sequence homologs are cloned from cDNA and genomicDNA libraries by hybridization with probes, or isolated usingamplification techniques with oligonucleotide primers. For example, T2Rsequences are typically isolated from mammalian nucleic acid (genomic orcDNA) libraries by hybridizing with a nucleic acid probe, the sequenceof which can be derived from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, or SEQ ID NO:81. A suitable tissue from which RNA and cDNA forT2R family members can be isolated is tongue tissue, optionally tastebud tissues or individual taste cells.

Amplification techniques using primers can also be used to amplify andisolate T2R sequences from DNA or RNA. For example, degenerate primersencoding the following amino acid sequences can be used to amplify asequence of a T2R gene: SEQ ID NOS: 50, 51, 52, or 53 (see, e.g.,Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995)). Theseprimers can be used, e.g., to amplify either the full length sequence ora probe of one to several hundred nucleotides, which is then used toscreen a mammalian library for full-length T2R clones. In addition,degenerate primers encoding the following amino acid sequences can beused to amplify a sequence of an SF01 (GR01) gene: SEQ ID NOS:88, 89,90, or 91. As described above, such primers can be used to isolate afull length sequence, or a probe which can then be used to isolated afull length sequence, e.g., from a library.

Nucleic acids encoding T2R can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, or SEQ ID NO:80.

Polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to a T2R family member can be isolated using T2Rnucleic acid probes, and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone T2R family members and T2R family memberpolymorphic variants, alleles, and interspecies homologs, by detectingexpressed homologs immunologically with antisera or purified antibodiesmade against a T2R polypeptide, which also recognize and selectivelybind to the T2R homolog.

To make a cDNA library, one should choose a source that is rich in T2RmRNA, e.g., tongue tissue, or isolated taste buds. The mRNA is then madeinto cDNA using reverse transcriptase, ligated into a recombinantvector, and transfected into a recombinant host for propagation,screening and cloning. Methods for making and screening cDNA librariesare well known (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating T2R nucleic acid and its homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of T2Rgenes directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. Degenerate oligonucleotides can be designed to amplify T2Rfamily member homologs using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of T2R-encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of T2R family members can also be analyzed by techniquesknown in the art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, probing DNA microchip arrays,and the like. In one embodiment, high density oligonucleotide analysistechnology (e.g., GeneChip™) is used to identify homologs andpolymorphic variants of the GPCRs of the invention. In the case wherethe homologs being identified are linked to a known disease, they can beused with GeneChip™ as a diagnostic tool in detecting the disease in abiological sample, see, e.g., Gunthand et al., AIDS Res. Hum.Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759(1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart etal., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res.8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).

Synthetic oligonucleotides can be used to construct recombinant T2Rgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the T2R nucleic acid. The specificsubsequence is then ligated into an expression vector.

The nucleic acid encoding a T2R gene is typically cloned intointermediate vectors before transformation into prokaryotic oreukaryotic cells for replication and/or expression. These intermediatevectors are typically prokaryote vectors, e.g., plasmids, or shuttlevectors.

Optionally, nucleic acids encoding chimeric proteins comprising a T2Rpolypeptide or domains thereof can be made according to standardtechniques. For example, a domain such as a ligand binding domain, anextracellular domain, a transmembrane domain (e.g., one comprising seventransmembrane regions and corresponding extracellular and cytosolicloops), the transmembrane domain and a cytoplasmic domain, an activesite, a subunit association region, etc., can be covalently linked to aheterologous protein. For example, an extracellular domain can be linkedto a heterologous GPCR transmembrane domain, or a heterologous GPCRextracellular domain can be linked to a transmembrane domain. Otherheterologous proteins of choice include, e.g., green fluorescentprotein, β-gal, glutamate receptor, and the rhodopsin presequence.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene or nucleic acid, suchas those cDNAs encoding a T2R family member, one typically subclones theT2R sequence into an expression vector that contains a strong promoterto direct transcription, a transcription/translation terminator, and iffor a nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the T2R protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kitsfor such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available. In one embodiment,the eukaryotic expression vector is an adenoviral vector, anadeno-associated vector, or a retroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the T2R-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding a T2Rand signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence encoding a T2R may typically be linked to a cleavable signalpeptide sequence to promote secretion of the encoded protein by thetransformed cell. Such signal peptides would include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene, introns with functionalsplice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as neomycin, hymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a sequence encoding a T2Rfamily member under the direction of the polyhedrin promoter or otherstrong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of a T2Rprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressing aT2R gene.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe T2R family member, which is recovered from the culture usingstandard techniques identified below.

IV. Purification of T2R Polypeptides

Either naturally occurring or recombinant T2R polypeptides can bepurified for use in functional assays. Optionally, recombinant T2Rpolypeptides are purified. Naturally occurring T2R polypeptides arepurified, e.g., from mammalian tissue such as tongue tissue, and anyother source of a T2R homolog. Recombinant T2R polypeptides are purifiedfrom any suitable bacterial or eukaryotic expression system, e.g., CHOcells or insect cells.

T2R proteins may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant T2R familymembers are being purified. For example, proteins having establishedmolecular adhesion properties can be reversibly fused to the T2Rpolypeptide. With the appropriate ligand, a T2R can be selectivelyadsorbed to a purification column and then freed from the column in arelatively pure form. The fused protein is then removed by enzymaticactivity. Finally T2R proteins can be purified using immunoaffinitycolumns.

A. Purification of T2R Protein from Recombinant Cells

Recombinant proteins are expressed by transformed bacteria or eukaryoticcells such as CHO cells or insect cells in large amounts, typicallyafter promoter induction; but expression can be constitutive. Promoterinduction with IPTG is a one example of an inducible promoter system.Cells are grown according to standard procedures in the art. Fresh orfrozen cells are used for isolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of T2Rinclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. T2R polypeptides areseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

Alternatively, it is possible to purify T2R polypeptides from bacteriaperiplasm. After lysis of the bacteria, when a T2R protein is exportedinto the periplasm of the bacteria, the periplasmic fraction of thebacteria can be isolated by cold osmotic shock in addition to othermethods known to skill in the art. To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

B. Standard Protein Separation Techniques for Purifying T2R Polypeptides

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of a T2R protein can be used to isolated it fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

T2R proteins can also be separated from other proteins on the basis ofits size, net surface charge, hydrophobicity, and affinity for ligands.In addition, antibodies raised against proteins can be conjugated tocolumn matrices and the proteins immunopurified. All of these methodsare well known in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

V. Immunological Detection of T2R Polypeptides

In addition to the detection of T2R genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect T2R, e.g., to identify taste receptor cells and variants of T2Rfamily members. Immunoassays can be used to qualitatively orquantitatively analyze the T2R. A general overview of the applicabletechnology can be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988).

A. Antibodies to T2R Family Members

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with a T2R family member are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of T2R-comprising immunogens may be used to produce antibodiesspecifically reactive with a T2R family member. For example, arecombinant T2R protein, or an antigenic fragment thereof, is isolatedas described herein. Suitable antigenic regions include, e.g., theconserved motifs that are used to identify members of the T2R family andthe SF01 gene, i.e., SEQ ID NOS: SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84; SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87; SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, and SEQ ID NO:91. Recombinant protein can beexpressed in eukaryotic or prokaryotic cells as described above, andpurified as generally described above. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Alternatively, a synthetic peptide derived from thesequences disclosed herein and conjugated to a carrier protein can beused an immunogen. Naturally occurring protein may also be used eitherin pure or impure form. The product is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies may be generated, for subsequent use in immunoassays tomeasure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the T2R. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-T2R proteins,or even other T2R family members or other related proteins from otherorganisms, using a competitive binding immunoassay. Specific polyclonalantisera and monoclonal antibodies will usually bind with a K_(d) of atleast about 0.1 mM, more usually at least about 1 μM, optionally atleast about 0.1 μM or better, and optionally 0.01 μM or better.

Once T2R family member specific antibodies are available, individual T2Rproteins can be detected by a variety of immunoassay methods. For areview of immunological and immunoassay procedures, see Basic andClinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological Binding Assays

T2R proteins can be detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case a T2R family member or anantigenic subsequence thereof). The antibody (e.g., anti-T2R) may beproduced by any of a number of means well known to those of skill in theart and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled T2R polypeptide or alabeled anti-T2R antibody. Alternatively, the labeling agent may be athird moiety, such a secondary antibody, that specifically binds to theantibody/T2R complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, optionally from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting a T2R protein in a sample may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-T2R antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture the T2R protein present in the testsample. The T2R protein is thus immobilized is then bound by a labelingagent, such as a second T2R antibody bearing a label. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

Competitive Assay Formats

In competitive assays, the amount of T2R protein present in the sampleis measured indirectly by measuring the amount of a known, added(exogenous) T2R protein displaced (competed away) from an anti-T2Rantibody by the unknown T2R protein present in a sample. In onecompetitive assay, a known amount of T2R protein is added to a sampleand the sample is then contacted with an antibody that specificallybinds to the T2R. The amount of exogenous T2R protein bound to theantibody is inversely proportional to the concentration of T2R proteinpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. The amount of T2R proteinbound to the antibody may be determined either by measuring the amountof T2R protein present in a T2R/antibody complex, or alternatively bymeasuring the amount of remaining uncomplexed protein. The amount of T2Rprotein may be detected by providing a labeled T2R molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known T2R protein is immobilized on a solid substrate. Aknown amount of anti-T2R antibody is added to the sample, and the sampleis then contacted with the immobilized T2R. The amount of anti-T2Rantibody bound to the known immobilized T2R protein is inverselyproportional to the amount of T2R protein present in the sample. Again,the amount of immobilized antibody may be detected by detecting eitherthe immobilized fraction of antibody or the fraction of the antibodythat remains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59,SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, orSEQ ID NO:81 can be immobilized to a solid support. Proteins (e.g., T2Rproteins and homologs) are added to the assay that compete for bindingof the antisera to the immobilized antigen. The ability of the addedproteins to compete for binding of the antisera to the immobilizedprotein is compared to the ability of the T2R polypeptide encoded by SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81 tocompete with itself. The percent crossreactivity for the above proteinsis calculated, using standard calculations. Those antisera with lessthan 10% crossreactivity with each of the added proteins listed aboveare selected and pooled. The cross-reacting antibodies are optionallyremoved from the pooled antisera by immunoabsorption with the addedconsidered proteins, e.g., distantly related homologs. In addition,peptides representing the conserved motifs that are used to identifymembers of the T2R family and the SF01 gene can be used incross-reactivity determinations, i.e., SEQ ID NOS: SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84; SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87; SEQ IDNO:88, SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:91

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of a T2R familymember, to the immunogen protein (i.e., T2R protein encoded by SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73,SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81). In order tomake this comparison, the two proteins are each assayed at a wide rangeof concentrations and the amount of each protein required to inhibit 50%of the binding of the antisera to the immobilized protein is determined.If the amount of the second protein required to inhibit 50% of bindingis less than 10 times the amount of the protein encoded by SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81 that is required toinhibit 50% of binding, then the second protein is said to specificallybind to the polyclonal antibodies generated to a T2R immunogen.

Polyclonal antibodies that specifically bind to a particular member ofthe T2R family, e.g., SF01, can be make by subtracting outcross-reactive antibodies using other T2R family members.Species-specific polyclonal antibodies can be made in a similar way. Forexample, antibodies specific to human SF01 can be made by subtractingout antibodies that are cross-reactive with rat or mouse SF01.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of T2R protein in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind the T2R protein. The anti-T2R polypeptideantibodies specifically bind to the T2R polypeptide on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to the anti-T2Rantibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize a T2R protein,or secondary antibodies that recognize anti-T2R.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of T2R Family Members

A. Assays for T2R Protein Activity

T2R family members and their alleles and polymorphic variants areG-protein coupled receptors that participate in taste transduction. Theactivity of T2R polypeptides can be assessed using a variety of in vitroand in vivo assays to determine functional, chemical, and physicaleffects, e.g., measuring ligand binding (e.g., radioactive ligandbinding), second messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ionflux, phosphorylation levels, transcription levels, neurotransmitterlevels, and the like. Furthermore, such assays can be used to test forinhibitors and activators of T2R family members. Modulators can also begenetically altered versions of T2R receptors. Such modulators of tastetransduction activity are useful for customizing taste.

The T2R protein of the assay will be selected from a polypeptide havinga sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, or SEQ ID NO:80 or conservatively modified variant thereof.Alternatively, the T2R protein of the assay will be derived from aeukaryote and include an amino acid subsequence having amino acidsequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, or SEQ ID NO:91. Generally, the amino acid sequence identity willbe at least 60%, optionally at least 70% to 85%, optionally at least90-95%. Optionally, the polypeptide of the assays will comprise a domainof a T2R protein, such as an extracellular domain, transmembrane domain,cytoplasmic domain, ligand binding domain, subunit association domain,active site, and the like. Either the T2R protein or a domain thereofcan be covalently linked to a heterologous protein to create a chimericprotein used in the assays described herein.

Modulators of T2R receptor activity are tested using T2R polypeptides asdescribed above, either recombinant or naturally occurring. The proteincan be isolated, expressed in a cell, expressed in a membrane derivedfrom a cell, expressed in tissue or in an animal, either recombinant ornaturally occurring. For example, tongue slices, dissociated cells froma tongue, transformed cells, or membranes can b used. Modulation istested using one of the in vitro or in vivo assays described herein.Taste transduction can also be examined in vitro with soluble or solidstate reactions, using a full-length T2R-GPCR or a chimeric moleculesuch as an extracellular domain of a receptor covalently linked to aheterologous signal transduction domain, or a heterologous extracellulardomain covalently linked to the transmembrane and or cytoplasmic domainof a receptor. Furthermore, ligand-binding domains of the protein ofinterest can be used in vitro in soluble or solid state reactions toassay for ligand binding.

Ligand binding to a T2R protein, a domain, or chimeric protein can betested in solution, in a bilayer membrane, attached to a solid phase, ina lipid monolayer, or in vesicles. Binding of a modulator can be testedusing, e.g., changes in spectroscopic characteristics (e.g.,fluorescence, absorbance, refractive index) hydrodynamic (e.g., shape),chromatographic, or solubility properties.

Receptor-G-protein interactions can also be examined. For example,binding of the G-protein to the receptor or its release from thereceptor can be examined. For example, in the absence of GTP, anactivator will lead to the formation of a tight complex of a G protein(all three subunits) with the receptor. This complex can be detected ina variety of ways, as noted above. Such an assay can be modified tosearch for inhibitors. Add an activator to the receptor and G protein inthe absence of GTP, form a tight complex, and then screen for inhibitorsby looking at dissociation of the receptor-G protein complex. In thepresence of GTP, release of the alpha subunit of the G protein from theother two G protein subunits serves as a criterion of activation.

An activated or inhibited G-protein will in turn alter the properties oftarget enzymes, channels, and other effector proteins. The classicexamples are the activation of cGMP phosphodiesterase by transducin inthe visual system, adenylate cyclase by the stimulatory G-protein,phospholipase C by Gq and other cognate G proteins, and modulation ofdiverse channels by Gi and other G proteins. Downstream consequences canalso be examined such as generation of diacyl glycerol and IP3 byphospholipase C, and in turn, for calcium mobilization by IP3.

Activated GPCR receptors become substrates for kinases thatphosphorylate the C-terminal tail of the receptor (and possibly othersites as well). Thus, activators will promote the transfer of ³²P fromgamma-labeled GTP to the receptor, which can be assayed with ascintillation counter. The phosphorylation of the C-terminal tail willpromote the binding of arrestin-like proteins and will interfere withthe binding of G-proteins. The kinase/arrestin pathway plays a key rolein the desensitization of many GPCR receptors. For example, compoundsthat modulate the duration a taste receptor stays active would be usefulas a means of prolonging a desired taste or cutting off an unpleasantone. For a general review of GPCR signal transduction and methods ofassaying signal transduction, see, e.g., Methods in Enzymology, vols.237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature10:349:117-27 (1991); Bourne et al., Nature 348:125-32 (1990); Pitcheret al., Annu. Rev. Biochem. 67:653-92 (1998).

Samples or assays that are treated with a potential T2R proteininhibitor or activator are compared to control samples without the testcompound, to examine the extent of modulation. Control samples(untreated with activators or inhibitors) are assigned a relative T2Ractivity value of 100. Inhibition of a T2R protein is achieved when theT2R activity value relative to the control is about 90%, optionally 50%,optionally 25-0%. Activation of a T2R protein is achieved when the T2Ractivity value relative to the control is 110%, optionally 150%,200-500%, or 1000-2000%.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing a T2R protein. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques,e.g., the “cell-attached” mode, the “inside-out” mode, and the “wholecell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595(1997)). Whole cell currents are conveniently determined using thestandard methodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85(1981). Other known assays include: radiolabeled ion flux assays andfluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Gonzales &Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al., J. Pharmacol. Meth.25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70(1994)). Generally, the compounds to be tested are present in the rangefrom 1 pM to 100 mM.

The effects of the test compounds upon the function of the polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on the polypeptides of thisinvention. When the functional consequences are determined using intactcells or animals, one can also measure a variety of effects such astransmitter release, hormone release, transcriptional changes to bothknown and uncharacterized genetic markers (e.g., northern blots),changes in cell metabolism such as cell growth or pH changes, andchanges in intracellular second messengers such as Ca²⁺, IP3, cGMP, orcAMP.

Preferred assays for G-protein coupled receptors include cells that areloaded with ion or voltage sensitive dyes to report receptor activity.Assays for determining activity of such receptors can also use knownagonists and antagonists for other G-protein coupled receptors asnegative or positive controls to assess activity of tested compounds. Inassays for identifying modulatory compounds (e.g., agonists,antagonists), changes in the level of ions in the cytoplasm or membranevoltage will be monitored using an ion sensitive or membrane voltagefluorescent indicator, respectively. Among the ion-sensitive indicatorsand voltage probes that may be employed are those disclosed in theMolecular Probes 1997 Catalog. For G-protein coupled receptors,promiscuous G-proteins such as Gα15 and Gα16 can be used in the assay ofchoice (Wilkie et al., Proc. Nat.'l Acad. Sci. USA 88:10049-10053(1991)). Such promiscuous G-proteins allow coupling of a wide range ofreceptors.

Receptor activation typically initiates subsequent intracellular events,e.g., increases in second messengers such as IP3, which releasesintracellular stores of calcium ions. Activation of some G-proteincoupled receptors stimulates the formation of inositol triphosphate(IP3) through phospholipase C-mediated hydrolysis ofphosphatidylinositol (Berridge & Irvine, Nature 312:315-21 (1984)). IP3in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP3 can be used to assess G-protein coupledreceptor function. Cells expressing such G-protein coupled receptors mayexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it may be desirable although not necessary to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase. There are cyclic nucleotide-gated ionchannels, e.g., rod photoreceptor cell channels and olfactory neuronchannels that are permeable to cations upon activation by binding ofcAMP or cGMP (see, e.g., Altenhofen et al., Proc. Natl. Acad. Sci.U.S.A. 88:9868-9872 (1991) and Dhallan et al., Nature 347:184-187(1990)). In cases where activation of the receptor results in a decreasein cyclic nucleotide levels, it may be preferable to expose the cells toagents that increase intracellular cyclic nucleotide levels, e.g.,forskolin, prior to adding a receptor-activating compound to the cellsin the assay. Cells for this type of assay can be made byco-transfection of a host cell with DNA encoding a cyclicnucleotide-crated ion channel, GPCR phosphatase and DNA encoding areceptor (e.g., certain glutamate receptors, muscarinic acetylcholinereceptors, dopamine receptors, serotonin receptors, and the like),which, when activated, causes a change in cyclic nucleotide levels inthe cytoplasm.

In a preferred embodiment, T2R protein activity is measured byexpressing a T2R gene in a heterologous cell with a promiscuousG-protein that links the receptor to a phospholipase C signaltransduction pathway (see Offermanns & Simon, J. Biol. Chem.270:15175-15180 (1995)). Optionally the cell line is HEK-293 (which doesnot naturally express T2R genes) and the promiscuous G-protein is Gα15(Offermanns & Simon, supra). Modulation of taste transduction is assayedby measuring changes in intracellular Ca²⁺ levels, which change inresponse to modulation of the T2R signal transduction pathway viaadministration of a molecule that associates with a T2R protein. Changesin Ca²⁺ levels are optionally measured using fluorescent Ca²⁺ indicatordyes and fluorometric imaging.

In one embodiment, the changes in intracellular cAMP or cGMP can bemeasured using immunoassays. The method described in Offermanns & Simon,J. Biol. Chem. 270:15175-15180 (1995) may be used to determine the levelof cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp.Cell and Mol. Biol. 11: 159-164 (1994) may be used to determine thelevel of cGMP. Further, an assay kit for measuring cAMP and/or cGMP isdescribed in U.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with³H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates wereseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist to cpm in the presence of buffer control(which may or may not contain an agonist).

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on signal transduction. A host cellcontaining a T2R protein of interest is contacted with a test compoundfor a sufficient time to effect any interactions, and then the level ofgene expression is measured. The amount of time to effect suchinteractions may be empirically determined, such as by running a timecourse and measuring the level of transcription as a function of time.The amount of transcription may be measured by using any method known tothose of skill in the art to be suitable. For example, mRNA expressionof the protein of interest may be detected using northern blots or theirpolypeptide products may be identified using immunoassays.Alternatively, transcription based assays using reporter gene may beused as described in U.S. Pat. No. 5,436,128, herein incorporated byreference. The reporter genes can be, e.g., chloramphenicolacetyltransferase, luciferase, β-galactosidase and alkaline phosphatase.Furthermore, the protein of interest can be used as an indirect reportervia attachment to a second reporter such as green fluorescent protein(see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the protein of interest. Asubstantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of the protein of interest.

B. Modulators

The compounds tested as modulators of a T2R family member can be anysmall chemical compound, or a biological entity, such as a protein,sugar, nucleic acid or lipid. Alternatively, modulators can begenetically altered versions of a T2R gene. Typically, test compoundswill be small chemical molecules and peptides. Essentially any chemicalcompound can be used as a potential modulator or ligand in the assays ofthe invention, although most often compounds can be dissolved in aqueousor organic (especially DMSO-based) solutions are used. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). It will be appreciated that thereare many suppliers of chemical compounds, including Sigma (St. Louis,Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs, Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provide soluble assays using moleculessuch as a domain such as ligand binding domain, an extracellular domain,a transmembrane domain (e.g., one comprising seven transmembrane regionsand cytosolic loops), the transmembrane domain and a cytoplasmic domain,an active site, a subunit association region, etc.; a domain that iscovalently linked to a heterologous protein to create a chimericmolecule; a T2R protein; or a cell or tissue expressing a T2R protein,either naturally occurring or recombinant. In another embodiment, theinvention provides solid phase based in vitro assays in a highthroughput format, where the domain, chimeric molecule, T2R protein, orcell or tissue expressing the T2R is attached to a solid phasesubstrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100-about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 different compoundsis possible using the integrated systems of the invention. Morerecently, microfluidic approaches to reagent manipulation have beendeveloped.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage, e.g., viaa tag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the taste transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethylene glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

D. Computer-Based Assays

Yet another assay for compounds that modulate T2R protein activityinvolves computer assisted drug design, in which a computer system isused to generate a three-dimensional structure of a T2R protein based onthe structural information encoded by its amino acid sequence. The inputamino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands. These regionsare then used to identify ligands that bind to the protein.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a T2R polypeptide intothe computer system. The nucleotide sequence encoding the polypeptide,or the amino acid sequence thereof, is preferably selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59,SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, orSEQ ID NO:81; or SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID) NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, or SEQ ID NO:80, respectively, and conservatively modifiedversions thereof. The amino acid sequence represents the primarysequence or subsequence of the protein, which encodes the structuralinformation of the protein. At least 10 residues of the amino acidsequence (or a nucleotide sequence encoding 10 amino acids) are enteredinto the computer system from computer keyboards, computer readablesubstrates that include, but are not limited to, electronic storagemedia (e.g., magnetic diskettes, tapes, cartridges, and chips), opticalmedia (e.g., CD ROM), information distributed by internet sites, and byRAM. The three-dimensional structural model of the protein is thengenerated by the interaction of the amino acid sequence and the computersystem, using software known to those of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the T2R protein to identify ligands that bind to the protein.Binding affinity between the protein and ligands is determined usingenergy terms to determine which ligands have an enhanced probability ofbinding to the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of T2R genes. Such mutationscan be associated with disease states or genetic traits. As describedabove, GeneChip™ and related technology can also be used to screen formutations, polymorphic variants, alleles and interspecies homologs. Oncethe variants are identified, diagnostic assays can be used to identifypatients having such mutated genes. Identification of the mutated T2Rgenes involves receiving input of a first nucleic acid or amino acidsequence of a T2R gene selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73,SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, and SEQ ID NO:81; or SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70,SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ IDNO:80, respectively, and conservatively modified versions thereof. Thesequence is entered into the computer system as described above. Thefirst nucleic acid or amino acid sequence is then compared to a secondnucleic acid or amino acid sequence that has substantial identity to thefirst sequence. The second sequence is entered into the computer systemin the manner described above. Once the first and second sequences arecompared, nucleotide or amino acid differences between the sequences areidentified. Such sequences can represent allelic differences in variousT2R genes, and mutations associated with disease states and genetictraits.

VIII. Kits

T2R genes and their homologs are useful tools for identifying tastereceptor cells, for forensics and paternity determinations, and forexamining taste transduction. T2R family member-specific reagents thatspecifically hybridize to T2R nucleic acids, such as T2R probes andprimers, and T2R specific reagents that specifically bind to a T2Rprotein, e.g., T2R antibodies are used to examine taste cell expressionand taste transduction regulation.

Nucleic acid assays for the presence of DNA and RNA for a T2R familymember in a sample include numerous techniques are known to thoseskilled in the art, such as Southern analysis, northern analysis, dotblots, RNase protection, S1 analysis, amplification techniques such asPCR and LCR, and in situ hybridization. In in situ hybridization, forexample, the target nucleic acid is liberated from its cellularsurroundings in such as to be available for hybridization within thecell while preserving the cellular morphology for subsequentinterpretation and analysis. The following articles provide an overviewof the art of in situ hybridization: Singer et al., Biotechniques4:230-250 (1986); Haase et al., Methods in Virology, vol. VII, pp.189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach(Hames et al., eds. 1987). In addition, a T2R protein can be detectedwith the various immunoassay techniques described above. The test sampleis typically compared to both a positive control (e.g., a sampleexpressing a recombinant T2R protein) and a negative control.

The present invention also provides for kits for screening formodulators of T2R family members. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: T2R nucleic acids orproteins, reaction tubes, and instructions for testing T2R activity.Optionally, the kit contains a biologically active T2R receptor. A widevariety of kits and components can be prepared according to the presentinvention, depending upon the intended user of the kit and theparticular needs of the user.

IX. Administration and Pharmaceutical Compositions

Taste modulators can be administered directly to the mammalian subjectfor modulation of taste in vivo. Administration is by any of the routesnormally used for introducing a modulator compound into ultimate contactwith the tissue to be treated, optionally the tongue or mouth. The tastemodulators are administered in any suitable manner, optionally withpharmaceutically acceptable carriers. Suitable methods of administeringsuch modulators are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985)).

The taste modulators, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by orally, topically, intravenously,intraperitoneally, intravesically or intrathecally. Optionally, thecompositions are administered orally or nasally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Solutions and suspensions can be preparedfrom sterile powders, granules, and tablets of the kind previouslydescribed. The modulators can also be administered as part a of preparedfood or drug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The dose will be determined by the efficacy of theparticular taste modulators employed and the condition of the subject,as well as the body weight or surface area of the area to be treated.The size of the dose also will be determined by the existence, nature,and extent of any adverse side-effects that accompany the administrationof a particular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administeredin a physician may evaluate circulating plasma levels of the modulator,modulator toxicities, and the production of anti-modulator antibodies.In general, the dose equivalent of a modulator is from about 1 ng/kg to10 mg/kg for a typical subject.

For administration, taste modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Identification of Human SF01

Human psychophysical tasting studies have shown that humans can becategorized as tasters, non-tasters, and super-tasters for the bittersubstance PROP (Bartoshut et al., Physiol Behav 58:2994). The geneticlocus involved in PROP tasting has been mapped to human interval 5p15(Reed et al., 1999 Am. J Hum. Genet. 64). Using DNA sequences from thisgenomic area (using information provided by the National Center forBiotechnology Information; On the World Wide Web at ncbi.nim.nih.gov), acomputational analysis was performed to identify novel open readingframes (ORFs) in this interval. The identification of ORFs wasfacilitated using various programs such as ORF finder, genefinder,fgenesh, etc. (See, e.g., the website at dot.imgen.bcm.tmc.edu). AllORFs larger than 100 amino acids were compared against public databasesusing BLAST (see, e.g., the National Center for BiotechnologyInformation), and genes with sequences related to known GPCRs werechosen for further analysis. Candidate sequences were then analyzed forputative transmembrane regions using standard programs (See, e.g.,dot.imgen.bcm.tmc.edu), in particular 7 putative transmembrane segmentsas expected for a GPCR. In this way, the human SF01 (GR01) sequence wasidentified, as shown in FIG. 1 and SEQ ID NOS: 35 and 36. The human SF01(GR01) gene maps to genomic region 5p15.

Example II Identification of the T2R Gene Family

To identify additional T2R genes, sequence databases were searched forsequences homologous to the human SF01 sequence. Using this screeningparadigm, a novel family of GPCRs was identified that includes twogenomic clusters of 4 and 9 genes, as well as a number of single loci(see, FIG. 1). A dendogram of various T2R family members is shown asFIG. 2.

The two gene clusters were mapped to human regions 12p13 and 7q31,respectively. Using the Jackson laboratory databases of mouse genetics(see, e.g., the website at informatics.jax.org/), and the human/mousehomology maps from the National Center for Biotechnology Information(NCBI)(On the World Wide Web at ncbi.nlm.nih.gov/Homology/), the T2Rcluster at 12p13 was found to correspond to a cluster of bitter-tastingloci in mice. This chromosomal interval has been proposed to includegenes involved in the detection of various bitter substances, includingsucrose octaacetate (soa), ruffinose acetate (roa), cycloheximide (cyx),and quinine (qui), and to be tightly linked to Prp on mouse chromosome 6(Lush et al., Genet. Res. 66:167-174 (1995)). It has been discoveredthat the T2R cluster at 12p13 is syntenic with this area of mousechromosome 6, and that it contains Prp.

Example III Isolation of Rat SF01

In order to isolate rodent homologs of the human T2R gene familymembers, a rat circumvallate cDNA library was screened for relatedsequences using low stringency hybridization conditions (7×SSC, 54° C.).Positive clones were picked, rescreened, and sequenced using automateddideoxy sequencing methods. The nucleotide and amino acid sequence of arat homolog of human SF01 (GR01) is shown as SEQ ID NOS:1 and 2,respectively.

Example IV Taste Cell Specific Expression of Human T2R Genes

The expression of human T2R genes in taste cells was determined in twoways: (1) PCR of taste cDNA using primers to T2R family members, and (2)screening of taste cDNA libraries for T2R family members using standardtechniques known to those of skill in the art.

Example V Creation of Knockout Mice for mT2R5

Bitter taste mediates aversive behaviors to protect animals against theingestion of noxious and toxic substances. T2R receptors have beenstrongly associated with bitter taste based on three criteria: (1) T2R5are genetically linked to loci controlling bitter taste responses inmice and humans (see Adler et al.). (2) Members of the T2R familyfunction in cell-based assays as receptors for bitter tastants(mT2R5-cycloheximide, hT2R16-sialycylin; rT2R10-strychnine;mT2R8-denatonium; -and hT2R4-denatonium; see PCT/US00/24821, publishedad WO 01/18050, herein incorporated by reference in its entirety). (3)genetic polymorphisms in T2R5 are associated with differences in tasteperception (e.g. cycloheximide taster and non-taste mice). To confirmthat T2R5 indeed functions in vivo as a validated bitter taste receptorwe generated KO animals for T2R5, a receptor for cycloheximide (seeChandrashekar et al.)

Creation of Knockout

Overlap PCR was used to replace the starting codon of the mT2R5 genewith the reverse tet-transactivator gene (rtTA). An internal Sal I sitewas used to ligate a lox flanked cassette containing the neomycinresistance to the 3′ end of the rtTA gene. The six kb 5′ arm was ligatedusing a unique Asc I site, and the two kb 3′ arm was ligated using aunique Pme I site.

129/SvJ ES cells were electroporated with the targeting DNA and selectedby G418. Positive clones were determined by Southern Blot. A crerecombinase construct was electroporated to remove the lox cassettecontaining neo, and positives confirmed by PCR across the lox junction.These clones were then injected into a C57 embryo and implanted. A totalof 5 chimaeres were mated to C57 mice, and agouti founders interbred togenerate homozygous knockout mice.

The nucleotide and amino acid sequences of the mT2R5 gene are asfollows:

(SEQ ID NO: 19) MLSAAEGILLSIATVEAGLGVLGNTFIALVNCMDWAKNNKLSMTGFLLIGLATSRIFIVWLLTLDAYAKLFYPSKYFSSSLIEIISYIWMTVNHLTVWFATSLSIFYFLKIANFSDCVFLWLKRRTDKAFVFLLGCLLTSWVISFSFVVKVMKDGKVNHRNRTSEMYWEKRQFTINYVFLNIGVISLFMMTLTACFLLIMSLWRHSRQMQSGVSGFRDLNTEAHVKAIKFLISFIILFVLYFIGVSIEIICIFIPENKLLFIFGFTTASIYPCCHSFILILSNSQLKQAFVKVLQGLKFF (SEQ ID NO: 30)ATGCTGAGTGCGGCAGAAGGCATCCTCCTTTCCATTGCAACTGTTGAAGCTGGGCTGGGAGTTTTAGGGAACACATTTATTGCACTGGTAAACTGCATGGACTGGGCCAAGAACAATAAGCTTTCTATGACTGGCTTCCTTCTCATCGGCTTAGCAACTTCCAGGATTTTTATTGTGTGGCTATTAACTTTAGATGCATATGCAAAGCTATTCTATCCAAGTAAGTATTTTTCTAGTAGTCTGATTGAAATCATCTCTTATATATGGATGACTGTGAATCACCTGACTGTCTGGTTTGCCACCAGCCTAAGCATCTTCTATTTCCTGAAGATAGCCAATTTTTCCGACTGTGTATTTCTCTGGTTGAAGAGGAGAACTGATAAAGCTTTTGTTTTTCTCTTGGGGTGTTTGCTAACTTCATGGGTAATCTCCTTCTCATTTGTTGTGAAGGTGATGAAGGACGGTAAAGTGAATCATAGAAACAGGACCTCGGAGATGTACTGGGAGAAAAGGCAATTCACTATTAACTACGTTTTCCTCAATATTGGAGTCATTTCTCTCTTTATGATGACCTTAACTGCATGTTTCTTGTTAATTATGTCACTTTGGAGACACAGCAGGCAGATGCAGTCTGGTGTTTCAGGATTCAGAGACCTCAACACAGAAGCTCATGTGAAAGCCATAAAATTTTTAATTTCATTTATCATCCTTTTCGTCTTGTATTTTATAGGTGTTTCAATAGAAATTATCTGCATATTTATACCAGAAAACAAACTGCTATTTATTTTTGGTTTCACAACTGCATCCATATATCCTTGCTGTCACTCATTTATTCTAATTCTATCTAACAGCCAGCTAAAGCAAGCCTTTGTAAAGGTACTGCAAGGATTAAAGTTCTTT TAG

Two-Bottle Assay

After weaning, two mice were placed in a cage with two bottles of water.After a week of training, one bottle was replaced by a tastant and thebottles weighed. After 24 hours, the left-right positions of the bottleswere switched. After 48 hours, the bottles were again weighed and thedifference noted. Preference is calculated as the percent of tastantconsumed over the total amount of liquid consumed. A version iscalculated as preference ratios.

1. An isolated nucleic acid encoding a T2R family taste transductionG-protein coupled receptor, wherein the receptor is expressed in a tastecell, the receptor comprising a polypeptide having greater than 95%amino acid sequence identity to SEQ ID NO:1.
 2. The isolated nucleicacid of claim 1, wherein the nucleic acid encodes a receptor that hasG-protein coupled receptor activity.
 3. The isolated nucleic acid ofclaim 1, wherein the nucleic acid encodes a receptor comprising an aminoacid sequence of SEQ ID NO:1.
 4. The isolated nucleic acid sequence ofclaim 1, wherein the nucleic acid comprises a nucleotide sequence of SEQID NO:2.
 5. The isolated nucleic acid of claim 1, wherein the nucleicacid is from a rat or a mouse.
 6. An expression vector comprising thenucleic acid of claim
 1. 7. An isolated cell comprising the vector ofclaim 6.